SUMMARY Survival of neuronal cells in response to various acute and chronic environmental stresses including infection, inflammation, and substance abuse depends on a cascade of biochemical reactions and signaling events to maintain energy production for cellular survival. Similar to HIV-1 infection, cocaine targets the brain and elicits a range of toxicities in multiple cell types, especially neurons. Previous findings suggest that chronic cocaine exposure and HIV-1 infection may synergize to exacerbate neuronal cell injury and death to a greater extent than either agent alone. As survival of neurons is highly dependent on the integrity and functionality of mitochondria, it is hypothesized that dysregulation of oxidative phosphorylation and impaired mitochondria function resulting from chronic cocaine use in the context of HIV-1 plays a critical role in the events leading to brain cell damage. Supporting this notion, we found that treating neuronal cells with cocaine decreases mitochondrial ATP production, an effect that was remarkably enhanced in the presence of Tat, a toxic HIV-1 protein found in blood and cerebrospinal fluid of HIV-1+ patients. Also, cocaine and HIV-1 Tat cooperatively suppressed mitochondrial function, seen as a decrease in membrane potential, and enhanced mitochondrial- dependent cell death. In addition, we found that Tat damages neuronal mitochondrial DNA (mDNA) by elevating ROS production, inhibiting antioxidant defenses, and impairing the base-excising mDNA repair enzyme OGG1, causing the accumulation of somatic mutations in mDNA. Such combinatorial action of HIV-1 Tat and cocaine also affected mitochondrial content/biogenesis by modulating mitophagy, via pathways involving BAG3 and the PINK1, parkin/HSP70 signaling axis. Together, these findings support our novel working model, in which cocaine and HIV-1 Tat act synergistically to promote mitochondrial dysfunction, diminishing ATP production and enhancing neuronal cell death. This project will leverage the expertise of all participants of this program project in cocaine research, mitochondrial biology, and HIV-1 neuropathogenesis, though concerted in vitro and in vivo studies to decipher the underlying molecular mechanisms responsible for the augmented neuropathological effects of cocaine abuse in the setting of HIV-1 infection.